Abstract

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

Pathology of tibialis anterior (TA) muscle of 4 week-old Mtm1 mutant mice. (A) Weight of wild-type (WT) and Mtm1/HSA [mKO (muscle-specific knockout)] TA muscles (n = 4 and 8 muscles for WT and mKO, respectively). Note the important reduction of muscle mass in mutant mice (*P < 0.001). (B) Area of TA myofibers. The curve represents the percentage of muscle fibers per area group (myofiber areas were divided into 20 groups, n = 496 and 771 for WT and mKO fibers, respectively, P < 0.001). The curve is shifted to the left in mKO animals indicating a general decrease of myofiber areas. (C) Hematoxylin and eosin (left panels, HE, magnification ×400) and nicotimanide adenine dinucleotide tetrazolium reductase (NADH-TR) (right panels, magnification ×200) staining of TA cross-sections from WT (top) and mKO (bottom) mice at 4 weeks of age. Note the presence of very small myofibers (arrow) and nuclei beneath the sarcolemma. Mitochondrial oxidative staining is often distributed as a ring at the periphery of the muscle fibers.

Myotubularin replacement leads to increased muscle volume. (A) Expression of myotubularin in muscle-specific knockout (mKO) muscle after rAAV2/1 transduction. phosphate-buffered saline (PBS) and adeno-associated virus (AAV)-treated mutant tibialis anterior (TA) cross-sections (mKO-PBS and mKO-AAV) were immuno-labeled with a rabbit polyclonal antibody against myotubularin. The antibody labels aspecifically nuclei in mutant muscle, whereas it detects myotubularin throughout the fiber with reinforcement in the sarcolemmal region 4 weeks after rAAV2/1-CMV-Mtm1 vector transfer (magnification ×400). (B) Weight of mutant TA muscle after myotubularin re-expression. The weight of isolated TA was measured 4 weeks after viral transduction (mKO-AAV) and compared with PBS-injected wild-type (WT) and mKO muscles (n = 6 for WT-PBS and mKO-AAV, n = 7 for mKO-PBS). The weight is lower by about four times in mutant animals (mKO-PBS, *P = 2 × 10−6), but significantly increased after rAAV (recombinant AAV) transfer (*P = 2 × 10−5). (C) Evaluation of mean myofiber area 4 weeks after intramuscular injection of rAAV2/1-CMV-Mtm1 vector. The size of Mtm1-deficient TA muscle fibers is smaller than WT animals (n = 473 from five mice and n = 1555 from four mice for WT-PBS and mKO-PBS, respectively, *P < 0.001). Viral transduction leads to a strong increase in myofiber area (n = 636 from four mice for mKO-AAV, *P < 0,001). (D) Distribution of muscle fibers from TA according to their size. The areas were subdivided into 20 subgroups ranging from the smallest to the biggest area, the interval between each subgroup corresponds to 82 µm2. The curve represents the percentage of fibers that belongs to each of the subgroups according to genotype and treatment. Note the strong reduction of very small fibers in mKO-AAV muscle.

Recovery of the contractile force in Mtm1-deficient skeletal muscle. (A) Extensor digitorum longus (EDL) and tibialis anterior (TA) muscle cross-sections 4 weeks after viral injection. The photographs illustrate the important increase in the volume of both mutant muscles. The bars represent 1 mm. (B) The histogram represents the isometric contractile force (mean ± SD) of isolated EDL and TA normalized by muscle length. Strength was also measured 4 weeks after injection. Results show a significant decrease in muscle force between wild-type and mKO animals at 8 weeks of age (*P < 0.001), whereas an important recovery of strength is observed after myotubularin treatment (force is about five times higher in treated versus untreated muscles, P-value between mKO-PBS (muscle-specific knockout-phosphate-buffered saline) and mKO-AAV (muscle-specific knockout-adeno-associated virus) is P = 0.0006 for both muscles).

Effect of overexpression of myotubularin in skeletal muscle. (A) Presence of needle-like structures (left panel, arrows) and vacuoles (right panel) in wild-type muscle overexpressing myotubularin. Semithin cross-sections of tibialis anterior were stained with toluidine blue (×630, left and ×1000, right). (B) Electron micrographs illustrate the nature of myotubularin-induced needle-like structures (left and middle panels). They resemble myelin-like structures and are occasionally associated with honey combs-like structures (arrow, middle panel). Note that the basal lamina (arrowhead) is not included. Vacuoles often contain degenerative membrane aggregates (arrow, right panel). Occasional authophagic vacuoles are also present (arrowhead). Bars represent 2 µm in the left and right panels and 400 nm in the middle panel. (C) Immunogold detection with an anti-myotubularin antibody shows labeling of the sarcolemma (arrowheads) and an intense signal on the membrane aggregates. Bars indicate 200 nm. (D) Left panel: central section of an electron tomogram from a cellular region containing such a membrane assembly. Membrane sheets clearly appear as being composed of two bilayers. Vesicular structures can be found attached to the membrane sheets (arrow). Central panel: section through the membrane assembly normal to the section plane as indicated by the dotted line in the left panel. This cross-section demonstrates that the lipid assemblies are flat layers and not tubular structures. Right panel: surface representation of the lipid layers which evidences the twisted nature of the close to parallel lipid assemblies. The bar represents 200 nm in the left and central panels and 125 nm in the right panel.